Lactic acid has wide industrial applicability including uses in chemical processing and synthesis, cosmetics, pharmaceuticals, plastics, and food production. Most industrial scale processes for making lactic acid are fermentation processes. Various lactic acid-producing bacteria have been used in those fermentation processes.
Recent research has investigated the use of recombinant yeast strains in lactic acid fermentation processes. Recombinant yeast potentially can provide several advantages over bacterial fermentations. Some yeast strains are more resistant to higher temperatures. This potentially allows for higher temperature fermentations, which can translate to faster rates of fermentations. Better resistance to high temperature can make it easier to purge a fermentation medium of contaminating microbes, as the medium can simply be heated to a temperature that the desired species can tolerate but at which the unwanted species die off. Lactic acid-producing bacteria such as Lactobacilli require a complex fermentation medium in order to produce efficiently. The complexity of the fermentation medium increases raw material costs and makes it more difficult and expensive to separate the lactic acid from the medium. Using recombinant yeast offers the possibility of reducing costs by using a simplified fermentation medium.
In addition, some yeast strains are more tolerant of reduced pH conditions that are lactic acid-producing bacteria. This is potentially a very important characteristic, as the pH of the fermentation medium will naturally drop as lactic acid is produced. In conventional processes, it is necessary to buffer the medium to a pH of about 5-8 with a base such as calcium hydroxide or calcium carbonate. This neutralizes the acid to form a lactate salt. This lactate salt must be split in a subsequent step to recover the lactate in the desired acid form. The need to buffer the fermentation medium therefore leads to significant additional costs for added raw materials (buffering agents and typically sulfuric acid to split the lactate salt), additional processing steps (to regenerate the free acid) and disposal of wastes (most often calcium carbonate that is generated in the salt-splitting step). These expenses can be reduced significantly if the fermentation can be conducted at reduced pH. The successful development of a lactic acid-producing strain that can tolerate reduced pH media is therefore greatly desirable.
Porro and coworkers have attempted to engineer a lactic-acid producing yeast by inserting an exogenous LDH (lactate dehydrogenase) gene into yeast cells from the species S. cerevisiae, K. lactic, T. delbrueckii and Z. bailii, and disrupting the cell's natural pyruvate pathway. See Porro et al., “Development of metabolically engineered Saccharomyces cerevisiae cells for the production of lactic acid”, Biotechnol. Prog. 1995 May-June; 11(3): 294-8; Porro et al., “Replacement of a metabolic pathway for large-scale production of lactic acid from engineered yeasts”, App. Environ. Microbiol. 1999 September:65(9):4211-5; Bianchi et al., “Efficient homolactic fermentation by Kluyveromyces lactis strains defective in pyruvate utilization and transformed with the heterologous LDH gene”, App. Environ. Microbiol. 2001 December; 67(12)5621-5. Porro was able to produce a recombinant yeast that produces lactic acid, but the strains did not perform nearly well enough for implementation in a commercial process. To qualify for use in an industrial environment, the strain must generate good yields of lactic acid (i.e., high conversion of the substrate to lactic acid) and high productivity (i.e., rapid metabolism of the substrate to lactic acid). The yeast preferably is able to tolerate a medium having a high titer of lactic acid.
More recently, Rajgarhia and coworkers have created recombinant yeast that exhibit higher yields and productivities than those of Porro. See, for example, WO 00/71738, WO 02/42471 and PCT/US02/16223. However, it is desirable to provide a recombinant yeast in which yields and/or productivities are even further improved. In particular, it is desirable to provide a recombinant yeast strain that produces lactic acid at good yields and productivities under the anaerobic and/or microaerobic conditions that favor lactic acid production.